Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Propionate reinforces epithelial identity and reduces aggressiveness of lung carcinoma.

The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability.

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.

Full Separation and Sequencing of Isomeric Proteoforms in the Middle-Down Mass Range Using Cyclic Ion Mobility and Electron Capture Dissociation.

Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.

Does the presence of ground state complex between a PR-10 protein and a sensitizer affect the mechanism of sensitized photo-oxidation?

The mechanisms of one-electron protein oxidation are complicated and still not well-understood. In this work, we investigated the reaction of sensitized photo-oxidation using carboxybenzophenone (CB) as a sensitizer and a PR-10 protein (MtN13) as a quencher, which is intrinsically complicated due to the complex structure of the protein and multiple possibilities of CB attack. To predict and examine the possible reactions precisely, the 3D structure of the MtN13 protein was taken into account. Our crystallographic studies revealed a specific binding of the CB molecule in the protein's hydrophobic cavity, while mass spectrometry identified the amino acid residues (Met, Tyr, Asp and Phe) creating adducts with the sensitizer, thus indicating the sites of 3CB* quenching. In addition, protein aggregation was also observed. The detailed mechanisms of CB quenching by the MtN13 molecule were elucidated by an analysis of transient products by means of time-resolved spectroscopy. The investigation of the transient and stable products formed during the protein photo-oxidation was based on the data obtained from HPLC-MS analysis of model compounds, single amino acids and dipeptides. Our proposed mechanisms of sensitized protein photo-oxidation emphasize the role of a ground state complex between the protein and the sensitizer and indicate several new and specific products arising as a result of one-electron oxidation. Based on the analysis of the transient and stable products, we have demonstrated the influence of neighboring groups, especially in the case of Tyr oxidation, where the tyrosyl radical can be formed via a direct electron transfer from Tyr to CB* or via an intramolecular electron transfer from Tyr to Met radical cation Met > S●+ or thiyl radical CysS● from neighboring oxidized groups.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

Quantitative phosphoproteome analysis of Streptomyces coelicolor by immobilized zirconium (IV) affinity chromatography and mass spectrometry reveals novel regulated protein phosphorylation sites and sequence motifs.

Streptomycetes are multicellular gram-positive bacteria that produce many bioactive compounds, including antibiotics, antitumorals and immunosuppressors. The Streptomyces phosphoproteome remains largely uncharted even though protein phosphorylation at Ser/Thr/Tyr is known to modulate morphological differentiation and specialized metabolic processes. We here expand the S. coelicolor phosphoproteome by optimised immobilized zirconium (IV) affinity chromatography and mass spectrometry to identify phosphoproteins at the vegetative and sporulating stages. We mapped 361 phosphorylation sites (41% pSer, 56.2% pThr, 2.8% pTyr) and discovered four novel Thr phosphorylation motifs ("Kxxxx(pT)xxxxK", "DxE(pT)", "D(pT)" and "Exxxxx(pT)") in 351 phosphopeptides derived from 187 phosphoproteins. We identified 154 novel phosphoproteins, thereby almost doubling the number of experimentally verified Streptomyces phosphoproteins. Novel phosphoproteins included cell division proteins (FtsK, CrgA) and specialized metabolism regulators (ArgR, AfsR, CutR and HrcA) that were differentially phosphorylated in the vegetative and in the antibiotic producing sporulating stages. Phosphoproteins involved in primary metabolism included 27 novel ribosomal proteins that were phosphorylated during the vegetative stage. Phosphorylation of these proteins likely participate in the intricate and incompletely understood regulation of Streptomyces development and secondary metabolism. We conclude that Zr(IV)-IMAC is an efficient and sensitive method to study protein phosphorylation and regulation in bacteria and enhance our understanding of bacterial signalling. SIGNIFICANCE: Two thirds of the secondary metabolites used in clinic, especially antibiotics, were discovered in Streptomyces strains. Antibiotic resistance became one of the major challenges in clinic, and new antibiotics are urgently required in clinic. Next-generation sequencing analyses revealed that streptomycetes harbour many cryptic secondary metabolite pathways, i.e. pathways not expressed in the laboratory. Secondary metabolism is tightly connected with hypha differentiation and sporulation, and understanding Streptomyces differentiation is one of the main challenges in industrial microbiology, in order to activate the expression of cryptic pathways in the laboratory. Protein phosphorylation at Ser/Thr/Tyr modulates development and secondary metabolism, but the Streptomyces phosphoproteome is still largely uncharted. Previous S. coelicolor phosphoproteomic studies used TiO2 affinity enrichment and LC-MS/MS identifying a total of 184 Streptomyces phosphoproteins. Here, we used by first time zirconium (IV) affinity chromatography and mass spectrometry, identifying 186 S. coelicolor phosphoproteins. Most of these phosphoproteins (154) were not identified in previous phosphoproteomic studies using TiO2 affinity enrichment. Thereby we almost doubling the number of experimentally verified Streptomyces phosphoproteins. Zr(IV)-IMAC affinity chromatography also worked in E. coli, allowing the identification of phosphoproteins that were not identified by TiO2 affinity chromatography. We conclude that Zr(IV)-IMAC is an efficient and sensitive method for studies of protein phosphorylation and regulation in bacteria to enhance our understanding of bacterial signalling networks. Moreover, the new Streptomyces phosphoproteins identified will contribute to design further works to understand and modulate Streptomyces secondary metabolism activation.

FLASHIda enables intelligent data acquisition for top-down proteomics to boost proteoform identification counts.

The detailed analysis and structural characterization of proteoforms by top-down proteomics (TDP) has gained a lot of interest in biomedical research. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complexity of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. Here, we present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In an analysis of E. coli lysate, FLASHIda increases the number of unique proteoform level identifications from 800 to 1500 or generates a near-identical number of identifications in one third of the instrument time when compared to standard DDA mode. Furthermore, FLASHIda enables sensitive mapping of post-translational modifications and detection of chemical adducts. As a software extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.

The endocytic receptor uPARAP is a regulator of extracellular thrombospondin-1.

Thrombospondin-1 (TSP-1) is a matricellular protein with a multitude of functions in the pericellular and extracellular environment. We report a novel pathway for the regulation of extracellular TSP-1, governed by the endocytic collagen receptor, uPARAP (urokinase plasminogen activator receptor-associated protein; MRC2 gene product, also designated Endo180, CD280). First, using a novel proteomic approach for unbiased identification of ligands for endocytosis, we identify TSP-1 as a candidate ligand for specific uptake by uPARAP. We then show that uPARAP can efficiently internalize TSP-1 for lysosomal degradation, that this capability is not shared by other, closely related endocytic receptors and that uPARAP serves to regulate the extracellular levels of TSP-1 in vitro. Using wild type and uPARAP null mice, we also demonstrate uPARAP-mediated endocytosis of TSP-1 in dermal fibroblasts in vivo. Unlike other uPARAP ligands, the interaction with TSP-1 is sensitive to heparin and the responsible molecular motifs in uPARAP are overlapping, but not identical with those governing the interaction with collagens. Finally, we show that uPARAP can also mediate the endocytosis of TSP-2, a thrombospondin closely related to TSP-1, but not the more distantly related members of the same protein family, TSP-3, -4 and -5. These findings indicate that the role of uPARAP in ECM remodeling is not limited to the uptake of collagen for degradation but also includes an orchestrator function in the regulation of thrombospondins with numerous downstream effects. This is likely to be an important factor in the physiological and pathological roles of uPARAP in bone biology, fibrosis and cancer. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD031272.

Real-world particulate matters induce lung toxicity in rats fed with a high-fat diet: Evidence of histone modifications.

Exposure to ambient particulate matters (PMs) has been associated with a variety of lung diseases, and high-fat diet (HFD) was reported to exacerbate PM-induced lung dysfunction. However, the underlying mechanisms for the combined effects of HFD and PM on lung functions remain poorly unraveled. By performing a comparative proteomic analysis, the current study investigated the global changes of histone post-translational modifications (PTMs) in rat lung exposed to long-term, real-world PMs. In result, after PM exposure the abundance of four individual histone PTMs (1 down-regulated and 3 up-regulated) and six combinatorial PTMs (1 down-regulated and 5 up-regulated) were significantly altered in HFD-fed rats while only one individual PTM was changed in rats with normal diet (ND) feeding. Histones H3K18ac, H4K8ac and H4K12ac were reported to be associated with DNA damage response, and we found that these PTMs were enhanced by PM in HFD-fed rats. Together with the elevated DNA damage levels in rat lungs following PM and HFD co-exposure, we demonstrate that PM exposure combined with HFD could induce lung injury through altering more histone modifications accompanied by DNA damage. Overall, these findings will augment our knowledge of the epigenetic mechanisms for pulmonary toxicity caused by ambient PM and HFD exposure.

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.

Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers.

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and ß-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase.

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

Histo-molecular differentiation of renal cancer subtypes by mass spectrometry imaging and rapid proteome profiling of formalin-fixed paraffin-embedded tumor tissue sections.

Pathology differentiation of renal cancer types is challenging due to tissue similarities or overlapping histological features of various tumor (sub) types. As assessment is often manually conducted outcomes can be prone to human error and therefore require high-level expertise and experience. Mass spectrometry can provide detailed histo-molecular information on tissue and is becoming increasingly popular in clinical settings. Spatially resolving technologies such as mass spectrometry imaging and quantitative microproteomics profiling in combination with machine learning approaches provide promising tools for automated tumor classification of clinical tissue sections. In this proof of concept study we used MALDI-MS imaging (MSI) and rapid LC-MS/MS-based microproteomics technologies (15 min/sample) to analyze formalin-fixed paraffin embedded (FFPE) tissue sections and classify renal oncocytoma (RO, n = 11), clear cell renal cell carcinoma (ccRCC, n = 12) and chromophobe renal cell carcinoma (ChRCC, n = 5). Both methods were able to distinguish ccRCC, RO and ChRCC in cross-validation experiments. MSI correctly classified 87% of the patients whereas the rapid LC-MS/MS-based microproteomics approach correctly classified 100% of the patients. This strategy involving MSI and rapid proteome profiling by LC-MS/MS reveals molecular features of tumor sections and enables cancer subtype classification. Mass spectrometry provides a promising complementary approach to current pathological technologies for precise digitized diagnosis of diseases.

Combinatory Treatment of Canavanine and Arginine Deprivation Efficiently Targets Human Glioblastoma Cells via Pleiotropic Mechanisms.

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood-brain barrier.

Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFß mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2C134W hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2C134W and the TGFß pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.

PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia.

Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.

High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultrahigh vacuum (UHV) needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the high vacuum (HV) region. Either greatly ameliorates the pressure increases upon He or H2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.