Reprogramming neuroblastoma by diet-enhanced polyamine depletion.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

Clostridium scindens secretome suppresses virulence gene expression of Clostridioides difficile in a bile acid-independent manner.

Clostridioides difficile infection (CDI) is a major health concern and one of the leading causes of hospital-acquired diarrhea in many countries. C. difficile infection is challenging to treat as C. difficile is resistant to multiple antibiotics. Alternative solutions are needed as conventional treatment with broad-spectrum antibiotics often leads to recurrent CDI. Recent studies have shown that specific microbiota-based therapeutics such as bile acids (BAs) are promising approaches to treat CDI. Clostridium scindens encodes the bile acid-induced (bai) operon that carries out 7-alpha-dehydroxylation of liver-derived primary BAs to secondary BAs. This biotransformation is thought to increase the antibacterial effects of BAs on C. difficile. Here, we used an automated multistage fermentor to study the antibacterial actions of C. scindens and BAs on C. difficile in the presence/absence of a gut microbial community derived from healthy human donor fecal microbiota. We observed that C. scindens inhibited C. difficile growth when the medium was supplemented with primary BAs. Transcriptomic analysis indicated upregulation of C. scindens bai operon and suppressed expression of C. difficile exotoxins that mediate CDI. We also observed BA-independent antibacterial activity of the secretome from C. scindens cultured overnight in a medium without supplementary primary BAs, which suppressed growth and exotoxin expression in C. difficile mono-culture. Further investigation of the molecular basis of our observation could lead to a more specific treatment for CDI than current approaches. IMPORTANCE There is an urgent need for new approaches to replace the available treatment options against Clostridioides difficile infection (CDI). Our novel work reports a bile acid-independent reduction of C. difficile growth and virulence gene expression by the secretome of Clostridium scindens. This potential treatment combined with other antimicrobial strategies could facilitate the development of alternative therapies in anticipation of CDI and in turn reduce the risk of antimicrobial resistance.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

BACKGROUND: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. METHODS: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. RESULTS: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. CONCLUSIONS: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.

Performance Characteristics of Mass Spectrometry-Based Analytical Procedures for Quantitation of Nitrosamines in Pharmaceuticals: Insights from an Inter-laboratory Study.

With the discovery of carcinogenic nitrosamine impurities in pharmaceuticals in 2018 and subsequent regulatory requirements for risk assessment for nitrosamine formation during pharmaceutical manufacturing processes, storage or from contaminated supply chains, effective testing of nitrosamines has become essential to ensure the quality of drug substances and products. Mass spectrometry has been widely applied to detect and quantify trace amounts of nitrosamines in pharmaceuticals. As part of an effort by regulatory authorities to assess the measurement variation in the determination of nitrosamines, an inter-laboratory study was performed by the laboratories from six regulatory agencies with each of the participants using their own analytical procedures to determine the amounts of nitrosamines in a set of identical samples. The results demonstrated that accurate and precise quantitation of trace level nitrosamines can be achieved across multiple analytical procedures and provided insight into the performance characteristics of mass spectrometry-based analytical procedures in terms of accuracy, repeatability and reproducibility.

Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components.

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease.

Independent control of COVID-19 vaccines by EU Official Control Authority Batch Release: challenges, strengths and successes.

Vaccines have been a key tool in stemming the tide of the COVID-19 pandemic. The rapid development of effective vaccines against COVID-19, together with their regulatory approval and wide scale distribution has been achieved in an impressively short period thanks to the intense efforts of many. In parallel to vaccine development, the EU considered it important to prepare for the independent control of the COVID-19 vaccines, including testing, to help ensure that only vaccines that comply with the approved quality requirements reach the public and to help improve/increase public confidence in the vaccines. The existing EU Official Control Authority Batch Release (OCABR) system, co-ordinated by the European Directorate for the Quality of Medicines and HealthCare (EDQM), was able to effectively respond to the need, through rapid co-ordination, work-sharing, advance planning and early interaction with manufacturers, the Coalition for Epidemic Preparedness Innovation (CEPI) and regulatory authorities. The Official Medicines Control Laboratories (OMCLs) involved in the OCABR activity, using the strength of the established system in the OCABR network and adaptations to the crisis conditions, were ready to release the first COVID-19 vaccine batches, after protocol review and testing, at the time of the conditional marketing authorisation for each of the COVID-19 vaccines, with no delay for batches reaching the public. Thanks to the dedication of resources by the EU and national authorities as well as by the EDQM, this was done without impacting the release of the other vaccines and human blood and plasma derived medicinal products, essential for public health. Transparency and communication of practices were important factors to support reliance on the OCABR outcome in non-EU countries, with the goal to improve access to vaccines in Europe and beyond. An overview of the process, legal background, challenges and successes of OCABR for COVID-19 vaccines as well as a look at the international perspective and lessons learned is provided.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions.

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.

TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation.

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

International Regulatory Collaboration on the Analysis of Nitrosamines in Metformin-Containing Medicines.

Recalls of some batches of metformin have occurred due to the detection of N-nitrosodimethylamine (NDMA) in amounts above the acceptable intake (AI) of 96 ng per day. Prior to the recalls, an international regulatory laboratory network had been monitoring drugs for nitrosamine impurities with each laboratory independently developing and validating multiple analytical procedures to detect and measure nitrosamines in metformin drugs used in their jurisdictions. Here, we provide an overview of the analysis of metformin active pharmaceutical ingredients (APIs) and drug products with 1090 samples (875 finished dosage forms (FDFs) and 215 API samples) tested beginning in November of 2019 through July of 2020. Samples were obtained internationally by a variety of approaches, including purchased, received from firms via information requests or selected by regional regulatory authorities (either at wholesalers or during GMP inspections). Only one nitrosamine (NDMA) was detected and was only present in some batches of metformin products. For API samples, 213 out of 215 lots tested had no measurable level of NDMA. For FDF samples tested, the number of batches with NDMA above the AI amount for patient safety was 17.8% (156/875). Based on these data, although the presence of NDMA was of concern, 82.2% of the samples of metformin drug products tested met quality and safety standards for patients. Regulatory agencies continue to collaborate extensively and work with marketing authorization holders to understand root causes of nitrosamine formation and agree on corrective actions to mitigate the presence of NDMA in future metformin batches.

Heat shock induces premature transcript termination and reconfigures the human transcriptome.

The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation.

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.

Phosphorylation of the HP1ß hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency.

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.

A proteomic atlas of the neointima identifies novel druggable targets for preventive therapy.

AIMS: In-stent restenosis is a complication after coronary stenting associated with morbidity and mortality. Here, we sought to investigate the molecular processes underlying neointima formation and to identify new treatment and prevention targets. METHODS AND RESULTS: Neointima formation was induced by wire injury in mouse femoral arteries. High-accuracy proteomic measurement of single femoral arteries to a depth of about 5000 proteins revealed massive proteome remodelling, with more than half of all proteins exhibiting expression differences between injured and non-injured vessels. We observed major changes in the composition of the extracellular matrix and cell migration processes. Among the latter, we identified the classical transient receptor potential channel 6 (TRPC6) to drive neointima formation. While Trpc6-/- mice presented reduced neointima formation compared to wild-type mice (1.44 ± 0.39 vs. 2.16 ± 0.48, P = 0.01), activating or repressing TRPC6 in human vascular smooth muscle cells resulted in increased [vehicle 156.9 ± 15.8 vs. 1-oleoyl-2-acetyl-sn-glycerol 179.1 ± 8.07 (103 pixels), P = 0.01] or decreased migratory capacity [vehicle 130.0 ± 26.1 vs. SAR7334 111.4 ± 38.0 (103 pixels), P = 0.04], respectively. In a cohort of individuals with angiographic follow-up (n = 3068, males: 69.9%, age: 59 ± 11 years, follow-up 217.1 ± 156.4 days), homozygous carriers of a common genetic variant associated with elevated TRPC6 expression were at increased risk of restenosis after coronary stenting (adjusted odds ratio 1.49, 95% confidence interval 1.08-2.05; P = 0.01). CONCLUSIONS: Our study provides a proteomic atlas of the healthy and injured arterial wall that can be used to define novel factors for therapeutic targeting. We present TRPC6 as an actionable target to prevent neointima formation secondary to vascular injury and stent implantation.

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.

The glucocorticoid receptor recruits the COMPASS complex to regulate inflammatory transcription at macrophage enhancers.

Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis-regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.