Human Monocytes Engage an Alternative Inflammasome Pathway.

Interleukin-1ß (IL-1ß) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1ß independently of classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an "alternative inflammasome" that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K(+) efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1ß-mediated immune responses and immunopathology in humans.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Translational offsetting as a mode of estrogen receptor α-dependent regulation of gene expression.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel.

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics.

The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.

Cracking the genetic code with neural networks.

The genetic code is textbook scientific knowledge that was soundly established without resorting to Artificial Intelligence (AI). The goal of our study was to check whether a neural network could re-discover, on its own, the mapping links between codons and amino acids and build the complete deciphering dictionary upon presentation of transcripts proteins data training pairs. We compared different Deep Learning neural network architectures and estimated quantitatively the size of the required human transcriptomic training set to achieve the best possible accuracy in the codon-to-amino-acid mapping. We also investigated the effect of a codon embedding layer assessing the semantic similarity between codons on the rate of increase of the training accuracy. We further investigated the benefit of quantifying and using the unbalanced representations of amino acids within real human proteins for a faster deciphering of rare amino acids codons. Deep neural networks require huge amount of data to train them. Deciphering the genetic code by a neural network is no exception. A test accuracy of 100% and the unequivocal deciphering of rare codons such as the tryptophan codon or the stop codons require a training dataset of the order of 4-22 millions cumulated pairs of codons with their associated amino acids presented to the neural network over around 7-40 training epochs, depending on the architecture and settings. We confirm that the wide generic capacities and modularity of deep neural networks allow them to be customized easily to learn the deciphering task of the genetic code efficiently.

Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells.

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes.

In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.

Ribosome exit tunnel electrostatics.

The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in rabbit reticulocytes. Here we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show that a strong electrostatic screening is due to water molecules (not mobile ions) attracted to the ribosomal nucleic acid phosphate moieties buried in the immediate vicinity of the tunnel wall. We also show how the tunnel wall components and local ribosomal protein protrusions impact on the electrostatic potential profile and impede charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of -40% to +85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derive, at single-residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall, shape, and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, x-ray crystallography, physical chemistry, biomechanics, and synthetic and multiomics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control, and the role of the ribosome's mechanochemistry in the cotranslational protein folding.

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis.

The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.

Wobble uridine tRNA modification: a new vulnerability of refractory melanoma.

The enzymes catalysing the modification of the wobble uridine (U34) of tRNAs (U34-enzymes) play an important role in tumor development. We have recently demonstrated that the U34-enzymes are crucial in the survival of glycolytic melanoma cultures through a codon-specific regulation of HIF1α mRNA translation. Moreover, depletion of U34-enzymes resensitizes resistant melanoma to targeted therapy. These results indicate that targeting U34-enzymes represents a new therapeutic opportunity for melanoma patients.

Modeling Primary Human Monocytes with the Trans-Differentiation Cell Line BLaER1.

Monocytes and macrophages play a pivotal role in the induction and shaping of immune responses. Expressing a broad array of pattern recognition receptors (PRRs), monocytes and macrophages constitute an integral component of the innate branch of the immune system. Traditionally, the majority of innate immune sensing and signaling pathways have been studied in macrophages of the murine system. This is largely due to the fact that genetic loss-of-function studies are amenable in this species. On the other hand, human cell lines of the monocyte-macrophage cell lineage have been widely used to study myeloid cells in vitro. However, commonly utilized models (e.g., THP-1 cells) only mimic a limited spectrum of the immunobiology of primary human myeloid cells. Recently, we have explored the possibility to fill this gap with a human trans-differentiation cell culture system, in which lineage conversion from malignant B-lineage cells to monocytes/macrophages is caused by the inducible nuclear translocation of a C/EBPα transgene, BLaER1 cells. Using this model, we were able to characterize a novel inflammasome signaling entity that could not have been uncovered in the murine system or THP-1 cells. Here, we describe the handling of BLaER1 cells, providing a detailed protocol for their induced trans-differentiation. We also provide data to demonstrate the applicability of the BLaER1 monocyte/macrophage system to study phagocytosis and various PRR cascades in human cells.

tRNA Modification: Is Cancer Having a Wobble?

Translational control of protein synthesis supports tumor development and progression to metastasis. Wobble tRNA modifications are required during translation elongation and sustain proteome homeostasis. Recent work has highlighted the surprising upregulation of the wobble uridine 34 (U34) tRNA cascade in cancer, which underlies the specific requirement for this pathway in tumor development.

Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.

Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer.

Quantitative and qualitative changes in mRNA translation occur in tumor cells and support cancer progression and metastasis. Posttranscriptional modifications of transfer RNAs (tRNAs) at the wobble uridine 34 (U34) base are highly conserved and contribute to translation fidelity. Here, we show that ELP3 and CTU1/2, partner enzymes in U34 mcm5s2-tRNA modification, are up-regulated in human breast cancers and sustain metastasis. Elp3 genetic ablation strongly impaired invasion and metastasis formation in the PyMT model of invasive breast cancer. Mechanistically, ELP3 and CTU1/2 support cellular invasion through the translation of the oncoprotein DEK. As a result, DEK promotes the IRES-dependent translation of the proinvasive transcription factor LEF1. Consistently, a DEK mutant, whose codon composition is independent of U34 mcm5s2-tRNA modification, escapes the ELP3- and CTU1-dependent regulation and restores the IRES-dependent LEF1 expression. Our results demonstrate that the key role of U34 tRNA modification is to support specific translation during breast cancer progression and highlight a functional link between tRNA modification- and IRES-dependent translation during tumor cell invasion and metastasis.

Elp3 drives Wnt-dependent tumor initiation and regeneration in the intestine.

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.