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Simple Summary Disease progression while receiving treatment is a major problem in breast cancer. Mutations in the oestrogen receptor-α often lead to loss of drug activity, resulting in an inability of anti-oestrogens to stop cancer growth. There is an urgent need to establish which therapies can effectively eradicate cancer cells and also to understand how these therapies work. The aim of this study was to find compounds that diminish viability of breast cancer cells harbouring mutated oestrogen receptor-α. We discovered that cells, regardless of their oestrogen receptor-α mutational status, are vulnerable to the cell-cycle inhibitor ribociclib, which causes senescence accompanied by a decrease in sensitivity to various chemotherapies. Importantly, we found that viability of ribociclib-induced senescent cells is maintained by the EGFR signalling pathway, which may be therapeutically exploited. Abstract While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15–40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

Description, DNA helicases DHX36 and FANCJ constitute a previously unknown pathway that resolves G-quadruplex structures during DNA replication., G-quadruplex (or G4) structures form in guanine-rich DNA sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase composed of Cdc45, MCM2-7 and GINS (CMG) stalls at a leading strand G4 structure. Second, the DEAH-box helicase 36 (DHX36) mediates bypass of the CMG past the intact G4 structure, allowing approach of the leading strand to the G4. Third, G4 structure unwinding by the Fanconi anemia complementation group J helicase (FANCJ) enables DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring pathway robustness. This previously unknown genome maintenance pathway promotes faithful G4 replication, thereby avoiding genome instability.

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, of which rpnR was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the deletion strain ΔrpnR of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, ΔrpnR showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taken together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose.IMPORTANCEAspergillus niger secretes a high amount and diversity of industrially relevant enzymes into the culture medium. This makes this fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting expression of 36 predicted transcription factor genes. Here we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. This gene is thus an interesting target to improve enzyme production in A. niger.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC., Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, the authors use phosphoproteomics to define kinases with distinct activity profiles in TNBC, demonstrating their prognostic value as well as their utility for simplifying TNBC classification and designing drug regimens.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain post-translational modifications (PTMs), as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM and MRM(3) in the analysis of phosphorylation dynamics throughout the PI3K-mTOR and MAPK signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by post-analysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view on mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer. This article is protected by copyright. All rights reserved.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.

A crosslinking-mass spectrometry strategy, including a new proteome database search engine called XlinkX, enables the identification of inter- and intra-protein cross-links in cell lysates on a proteome-wide scale. We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)–tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,426 unique cross-links at a 5% FDR (2,013 intraprotein and 413 interprotein cross-links) or 1,822 cross-links at a 1% FDR (1,622 intraprotein and 200 interprotein cross-links), indicating the detection of 326 or 134 protein-protein interactions at 5% FDR or 1% FDR, respectively, in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.