Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression.

Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a pro-metastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression.

Nuclear TARBP2 Drives Oncogenic Dysregulation of RNA Splicing and Decay.

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages.

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.

Translation from unconventional 5' start sites drives tumour initiation.

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.

Publisher Correction: A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Transcriptomic characterization of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of ∼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.

Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors.

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

A crucial role for ß2 integrins in podosome formation, dynamics and Toll-like-receptor-signaled disassembly in dendritic cells.

The dynamic properties of podosomes, their ability to degrade the underlying matrix and their modulation by Toll-like receptor (TLR) signaling in dendritic cells (DCs) suggests they have an important role in migration. Integrins are thought to participate in formation and dynamics of podosomes but the multiplicity of integrins in podosomes has made this difficult to assess. We report that murine DCs that lack ß2 integrins fail to form podosomes. Re-expression of ß2 integrins restored podosomes but not when the membrane proximal or distal NPxF motifs, or when an intervening triplet of threonine residues were mutated. We show that ß2 integrins are remarkably long-lived in podosome clusters and form a persistent framework that hosts multiple actin-core-formation events at the same or adjacent sites. When ß2 integrin amino acid residues 745 or 756 were mutated from Ser to Ala, podosomes became resistant to dissolution mediated through TLR signaling. TLR signaling did not detectably modulate phosphorylation at these sites but mutation of either residue to phospho-mimetic Asp increased ß2 integrin turnover in podosomes, indicating that phosphorylation at one or both sites establishes permissive conditions for TLR-signaled podosome disassembly.

High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages.

Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicate that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1. Moreover, bone marrow derived macrophages phagosomes mature considerably faster by fusion with endosomes and the lysosome which we validated using fluorogenic phagocytic assays. We provide a valuable resource for researcher in the field and recommend careful use of the RAW 264.7 cell line when studying phagosome functions. All MS data have been deposited in the ProteomeXchange with identifier PXD001293 (http://proteomecentral.proteomexchange.org/dataset/PXD001293).

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome.

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

Enigmatic, ultrasmall, uncultivated Archaea.

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge-particularly for Archaea-that occur at relatively low abundance and in extreme environments. Ultrasmall cells (<500 nm in diameter) from lineages without cultivated representatives that branch near the crenarchaeal/euryarchaeal divide have been detected in a variety of acidic ecosystems. We reconstructed composite, near-complete approximately 1-Mb genomes for three lineages, referred to as ARMAN (archaeal Richmond Mine acidophilic nanoorganisms), from environmental samples and a biofilm filtrate. Genes of two lineages are among the smallest yet described, enabling a 10% higher coding density than found genomes of the same size, and there are noncontiguous genes. No biological function could be inferred for up to 45% of genes and no more than 63% of the predicted proteins could be assigned to a revised set of archaeal clusters of orthologous groups. Some core metabolic genes are more common in Crenarchaeota than Euryarchaeota, up to 21% of genes have the highest sequence identity to bacterial genes, and 12 belong to clusters of orthologous groups that were previously exclusive to bacteria. A small subset of 3D cryo-electron tomographic reconstructions clearly show penetration of the ARMAN cell wall and cytoplasmic membranes by protuberances extended from cells of the archaeal order Thermoplasmatales. Interspecies interactions, the presence of a unique internal tubular organelle [Comolli, et al. (2009) ISME J 3:159-167], and many genes previously only affiliated with Crenarchaea or Bacteria indicate extensive unique physiology in organisms that branched close to the time that Cren- and Euryarchaeotal lineages diverged.

Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

Global transcriptomic and proteomic responses of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation.

Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems-level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing cells of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation (FNL), as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially upregulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and is implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly upregulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally downregulated in response to FNL, and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition, while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and posttranslational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number of genes coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.