Targeting the Human 80S Ribosome in Cancer: From Structure to Function and Drug Design for Innovative Adjuvant Therapeutic Strategies

The human 80S ribosome is the cellular nucleoprotein nanomachine in charge of protein synthesis that is profoundly affected during cancer transformation by oncogenic proteins and provides cancerous proliferating cells with proteins and therefore biomass. Indeed, cancer is associated with an increase in ribosome biogenesis and mutations in several ribosomal proteins genes are found in ribosomopathies, which are congenital diseases that display an elevated risk of cancer. Ribosomes and their biogenesis therefore represent attractive anti-cancer targets and several strategies are being developed to identify efficient and specific drugs. Homoharringtonine (HHT) is the only direct ribosome inhibitor currently used in clinics for cancer treatments, although many classical chemotherapeutic drugs also appear to impact on protein synthesis. Here we review the role of the human ribosome as a medical target in cancer, and how functional and structural analysis combined with chemical synthesis of new inhibitors can synergize. The possible existence of oncoribosomes is also discussed. The emerging idea is that targeting the human ribosome could not only allow the interference with cancer cell addiction towards protein synthesis and possibly induce their death but may also be highly valuable to decrease the levels of oncogenic proteins that display a high turnover rate (MYC, MCL1). Cryo-electron microscopy (cryo-EM) is an advanced method that allows the visualization of human ribosome complexes with factors and bound inhibitors to improve our understanding of their functioning mechanisms mode. Cryo-EM structures could greatly assist the foundation phase of a novel drug-design strategy. One goal would be to identify new specific and active molecules targeting the ribosome in cancer such as derivatives of cycloheximide, a well-known ribosome inhibitor.

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Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness

eLife ◽

10.7554/elife.43002 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 25

Author(s):

Blake W Tye ◽

Nicoletta Commins ◽

Lillia V Ryazanova ◽

Martin Wühr ◽

Michael Springer ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Maximal Growth ◽

Proliferating Cells ◽

Cellular Processes ◽

Direct Contribution ◽

The Face ◽

Cellular Phenotypes

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

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The DHX33 RNA Helicase Promotes mRNA Translation Initiation

Molecular and Cellular Biology ◽

10.1128/mcb.00315-15 ◽

2015 ◽

Vol 35 (17) ◽

pp. 2918-2931 ◽

Cited By ~ 21

Author(s):

Yandong Zhang ◽

Jin You ◽

Xingshun Wang ◽

Jason Weber

Keyword(s):

Translation Initiation ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Mrna Translation ◽

Rna Helicase ◽

Rna Helicases ◽

80S Ribosome ◽

Protein Levels ◽

Cell Growth And Proliferation ◽

Rna Complexes

DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.

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O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0941 ◽

2010 ◽

Vol 21 (12) ◽

pp. 1922-1936 ◽

Cited By ~ 66

Author(s):

Quira Zeidan ◽

Zihao Wang ◽

Antonio De Maio ◽

Gerald W. Hart

Keyword(s):

Protein Synthesis ◽

Posttranslational Modifications ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Mammalian Target Of Rapamycin ◽

Nuclear Staining ◽

Mtor Signaling Pathway ◽

Translational Machinery ◽

Translation Factors ◽

Nucleolar Stress

Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.

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Roles of the mammalian target of rapamycin, mTOR, in controlling ribosome biogenesis and protein synthesis

Biochemical Society Transactions ◽

10.1042/bst20110682 ◽

2012 ◽

Vol 40 (1) ◽

pp. 168-172 ◽

Cited By ~ 50

Author(s):

Valentina Iadevaia ◽

Yilin Huo ◽

Ze Zhang ◽

Leonard J. Foster ◽

Christopher G. Proud

Keyword(s):

Protein Synthesis ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Kinase Inhibitors ◽

Mammalian Target Of Rapamycin ◽

Target Of Rapamycin ◽

Energy Status ◽

Stable Isotope Labelling ◽

Mtor Kinase ◽

The Impact

mTORC1 (mammalian target of rapamycin complex 1) is controlled by diverse signals (e.g. hormones, growth factors, nutrients and cellular energy status) and regulates a range of processes including anabolic metabolism, cell growth and cell division. We have studied the impact of inhibiting mTOR on protein synthesis in human cells. Partial inhibition of mTORC1 by rapamycin has only a limited impact on protein synthesis, but inhibiting mTOR kinase activity causes much greater inhibition of protein synthesis. Using a pulsed stable-isotope-labelling technique, we show that the rapamycin and mTOR (mammalian target of rapamycin) kinase inhibitors have differential effects on the synthesis of specific proteins. In particular, the synthesis of proteins encoded by mRNAs that have a 5′-terminal pyrimidine tract is strongly inhibited by mTOR kinase inhibitors. Many of these mRNAs encode ribosomal proteins. mTORC1 also promotes the synthesis of rRNA, although the mechanisms involved remain to be clarified. We found that mTORC1 also regulates the processing of the precursors of rRNA. mTORC1 thus co-ordinates several steps in ribosome biogenesis.

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Universality and non-universality of the growth law

10.1101/2021.12.02.471021 ◽

2021 ◽

Author(s):

Qirun Wang ◽

Jie Lin

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ribosomal Proteins ◽

Correlation Coefficients ◽

Growth Conditions ◽

Proliferating Cells ◽

Translation Speed ◽

Nutrient Quality ◽

Degradation Rates ◽

Mass Fractions

An approximately linear relationship between the fraction of ribosomal proteins in the proteome (ϕR) and the growth rate (μ) holds in proliferating cells when the nutrient quality changes, often referred to as a growth law. While a simple model assuming a constant translation speed of ribosomes without protein degradation can rationalize this growth law, real protein synthesis processes are more complex. This work proposes a general theoretical framework of protein synthesis, taking account of heterogeneous translation speeds among proteins and finite protein degradation. We introduce ribosome allocations as the fraction of active ribosomes producing certain proteins, with two correlation coefficients respectively quantifying the correlation between translation speeds and ribosome allocations, and between protein degradation rates and mass fractions. We prove that the growth law curve generally follows ϕR = (μ + c1)/(c2μ + c3) where c1, c2, and c3 are constants depending on the above correlation coefficients and the translation speed of ribosomal proteins. Our theoretical predictions of ϕR agree with existing data of Saccharomyces cerevisiae. We demonstrate that when different environments share similar correlation coefficients, the growth law curve is universal and up-bent relative to a linear line in slow-growth conditions, which appears valid for Escherichia coli. However, the growth law curve is non-universal and environmental-specific when the environments have significantly different correlation coefficients. Our theories allow us to estimate the translation speeds of ribosomal and non-ribosomal proteins based on the experimental growth law curves.

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Lenalidomide Induces A Ribosomal Stress Response In Multiple Myeloma (MM) Cells

Blood ◽

10.1182/blood.v122.21.3161.3161 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3161-3161

Author(s):

Jiri Slaby ◽

Ines Tagoug ◽

Paola Neri ◽

Justin Simms ◽

Jacquelyn Babich ◽

...

Keyword(s):

Stress Response ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

E3 Ligase ◽

Adaptor Protein ◽

Protein Stabilization ◽

Down Regulation ◽

80S Ribosome ◽

Recurrent Mutations ◽

Ribosomal Stress

Abstract Background Deregulation of ribosome biogenesis is associated with carcinogenesis and in MM two of the most common recurrent mutations (DIS3 and FAM46C) are implicated in ribosomal decay and translational control. Beside its role in the regulation of protein synthesis, the importance of ribosome biogenesis is underscored by the observation that the impairment of this process leads to a ribosomal stress response with induction of p53, inhibition of c-Myc and cell cycle arrest. These effects are mediated by the direct binding of ribosomal proteins (RPs), particularly RPL11 and RPL5 to Mdm2 and c-Myc. Immunomodulatory drugs (IMiDs) anti-MM effects require their binding to Cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex with an ensuing down-regulation of c-Myc and up-regulation of p21. In the present study, we delineated the mechanisms through which IMiDs mediate these effects in MM cells. Methods and Results In order to identify ubiquitylated substrates that are modified by lenalidomide (Len) treatment, we performed a ubiquitin-proteome pull-down using Tandem Ubiquitin Binding Entity (Lifesensors) coupled with quantitative mass-spectroscopy proteomics (iTRAQ) in OPM2 cells exposed to Len (10 μM). Several ribosomal proteins (RPs) - S25, S26, S20 & S28 - were increased in Len treated samples suggesting that the Cul4a-CRBN complex may regulate RPs stability and hence IMiDs binding to cereblon may trigger a ribosomal stress response. Immunoblot analysis of MM cells exposed to Len lead to a rapid decrease in c-Myc expression (within 30 min) that significantly preceded the downregulation of IRF4 consistent with an IRF4-independent mechanism for c-Myc down-regulation. Furthermore, Len treatment transiently stabilised and subsequently down-regulated MDM2 expression with up-regulation of p53 and its down-stream targets (p21, PUMA). Under ribosomal stress conditions, polysome-free RPs are released into the nucleoplasm where they bind Mdm2 and suppress its E3 ligase activity. Consistent with this effect, in Len treated cells MDM2 co-immunoprecipitated with RPL11 and RPL5 with p53 protein stabilization (no change in p53 mRNA). In addition, an increase in the interaction between RPL11 and c-Myc was also observed consistent with the reported role for RPL11 in the post-transcriptional regulation and suppression of c-Myc. Furthermore, treatment with Lenalidomide supressed ribosomal RNA (rRNA) transcription with inhibition of pre-rRNA (47S) processing. The examination of polysome fractions (sucrose gradients) in Len treated MM cells (OPM2 and MM1S) revealed a striking reduction of 60S and 80S as well as polysomes fractions, an effect similar to that Actinomycin D (5 nM), a known RNA PolI inhibitor and potent ribosome stress inducer. Furthermore Len treatment significant reduced RPL11 within the 60S and 80S ribosome fractions. Of note, CRBN knockdown abrogated all these effects suggesting that Len binding to CRBN is upstream of this ribosome stress response. In order to investigate whether the down-regulation of c-Myc is the primary event leading to the disruption of ribogenesis, we transiently and stably silenced RPL11 in MM cells. RPL11 silencing nearly fully protected MM cells from the effects of Len and importantly it completely reversed Len-induced Mdm2 E3 ligase inhibition with up-regulation of p53, p21 and suppression of c-Myc and IRF4. Conclusion Taken together our data indicate that treatment with lenalidomide suppresses ribogenesis and induces a ribosomal stress response downstream of Cul4a-CRBN but upstream of c-Myc suppression and p53 induction. These effects likely result from the IMiDs-induced modification of the Cul4a-CRBN ubiquitome and the regulation of RPs integration into ribosomal subunits. These findings also explain the observed clinical activity of IMiDs in other ribosomopathies like 5q- MDS and Diamond-Blackfan anemia Disclosures: No relevant conflicts of interest to declare.

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Control of ribosomal protein synthesis by the Microprocessor complex

Science Signaling ◽

10.1126/scisignal.abd2639 ◽

2021 ◽

Vol 14 (671) ◽

pp. eabd2639

Author(s):

Xuan Jiang ◽

Amit Prabhakar ◽

Stephanie M. Van der Voorn ◽

Prajakta Ghatpande ◽

Barbara Celona ◽

...

Keyword(s):

Protein Synthesis ◽

Ribosomal Protein ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Cellular Growth ◽

Ribosomal Protein Genes ◽

Erythroid Progenitors ◽

Microprocessor Complex ◽

Ribosomal Protein Synthesis

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human “ribosomopathies.” Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

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A risk-reward tradeoff of high ribosome production in proliferating cells

10.1101/458810 ◽

2018 ◽

Cited By ~ 2

Author(s):

Blake W. Tye ◽

Nicoletta Commins ◽

Michael Springer ◽

David Pincus ◽

L. Stirling Churchman

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Protein Assembly ◽

Ribosome Assembly ◽

Maximal Growth ◽

Proliferating Cells ◽

Cellular Processes ◽

Stress Response Pathway ◽

The Face ◽

Cellular Phenotypes

AbstractTo achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs), which are required to produce thousands of new ribosomes every minute. Although ribosomes are essential in all cells, disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we modeled these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result immediately in acute loss of proteostasis (protein folding homeostasis). Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes. In response, proteostasis genes are activated by an Hsf1-dependent stress response pathway that is required for recovery from r-protein assembly stress. Importantly, we show that exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. Our results highlight ribosome assembly as a linchpin of cellular homeostasis, representing a key proteostasis vulnerability for rapidly proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic conditions that generate orphan r-proteins.

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Shwachman — Diamond Syndrome: Modern Genetic Aspects of a Ribosomapathy

Doctor Ru ◽

10.31550/1727-2378-2020-19-10-33-36 ◽

2020 ◽

Vol 19 (10) ◽

pp. 33-36

Author(s):

M.G. Ipatova ◽

◽

Keyword(s):

High Risk ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Clinical Symptoms ◽

Genetic Disorders ◽

Exocrine Insufficiency ◽

Ribonucleoprotein Complexes ◽

Shwachman Diamond Syndrome ◽

Genetically Determined ◽

Risk Of Cancer

Objective of the Review: To analyse new DNAJC21, EFL1, SRP54 mutations causing ribosome biogenesis defects and presenting with clinical symptoms similar to the symptoms of Shwachman – Diamond syndrome (SDS). Key Points. SDS is a ribosomapathy and is characterised by pancreatic exocrine insufficiency, defective hematopoiesis, musculoskeletal anomalies, and a high risk of myelodysplastic syndrome and acute myeloid leukemia. About 90% of SDS patients have biallelic SBDS mutations. However, 10–20% of patients with a set of symptoms typical of SDS did not have any pathovars in SBDS gene; therefore, we searched for other candidate genes. In addition to SDS, genetic disorders associated with defected maturation, deficient structure or function of ribosomes and ribonucleoprotein complexes include Diamond – Blackfan anemia, cartilage and hair hypoplasy (McKusick type metaphyseal chondrodysplasia), congenital diskeratosis, 5q-syndrome, and others. These syndromes are similar to SDS. All these conditions are associated with medullary deficiency at least in one hematopoiesis chain. All five conditions are associated with a high risk of cancer. Conclusion. SDS is a genetically determined condition belonging to ribosomapathies. Ribosomapathies are caused by mutations in genes that participate in the synthesis of ribosomal proteins and factors, functioning at various stages of their assembly, and give origin to a number of clinical phenotypes, including haematological malignancies and cancer. In clinical practice, SDS is diagnosed on the basis of typical clinical symptoms and if pathogenic SBDS mutations are present. The issue whether SDS is a genetic heterogenetic ribosomapathy or a mutation of other genes causing defective ribosome synthesis and SDS-like symptoms, is disputable and requires further research. Keywords: Shwachman – Diamond syndrome, genes, SBDS, DNAJC21, EFL1, SRP54, ribosomapathy.

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Mosaic changes to the global transcriptome in response to inhibiting ribosome formation versus inhibition of ribosome function

10.1101/2020.10.15.341230 ◽

2020 ◽

Author(s):

Md Shamsuzzaman ◽

Nusrat Rahman ◽

Brian Gregory ◽

Vincent M Bruno ◽

Lasse Lindahl

Keyword(s):

Protein Synthesis ◽

Cell Fate ◽

Ribosomal Proteins ◽

Protein Transport ◽

Mitotic Cell ◽

Mitotic Cell Cycle ◽

Congenital Diseases ◽

External Stresses ◽

Global Transcriptome ◽

Ribosome Formation

AbstractCell fate is susceptible to several internal and external stresses. Stress resulting from mutations in genes for ribosomal proteins and assembly factors leads to many congenital diseases, collectively called ribosomopathies. Even though such mutations all depress the cell’s protein synthesis capacity, they are manifested in many different phenotypes. This prompted us to use Saccharomyces cerevisiae to explore whether reducing the protein synthesis capacity by different mechanisms result in the same or different changes to the global transcriptome. We have compared the transcriptome after abolishing the assembly of new ribosomes and inhibiting the translocation of ribosomes on the mRNA. Our results show that these alternate obstructions generate different mosaics of expression for several classes of genes, including genes for ribosomal proteins, mitotic cell cycle, cell wall synthesis, and protein transport.

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