Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells

Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome. Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A) cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs). We used pulse-SILAC MS (Boisvert et al., 2012), to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins. Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1) are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database (www.peptracker.com/epd). Conclusions: We present the first comprehensive analysis measuring how protein expression and protein turnover is affected by cell transformation, providing a detailed picture at the protein level of the consequences of activation of an oncogene

Identification of small molecule inhibitors of pre-mRNA splicing

Background: There is a need for new small molecule pre-mRNA splicing inhibitors as biotools. Results: High throughput screening resulted in the identification of small molecule splicing inhibitors that are active in vitro and in cells. Conclusion: New small molecules for studying pre-mRNA splicing in vitro and in cells are identified. Significance: Small drug-like molecules are identified that modulate splicing in vitro and in cells. Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed madrasin, i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells

Perturbation of Chromatin Structure Globally Affects Localization and Recruitment of Splicing Factors

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA

The Big Sibling of AU Mic: A Cold Dust-rich Debris Disk around CP-72 2713 in the β Pic Moving Group

Analyzing Spitzer and Herschel archival measurements we identified a hitherto unknown debris disk around the young K7/M0 star CP-72 2713. The system belongs to the 24Myr old $\beta$ Pic moving group. Our new 1.33mm continuum observation, obtained with the ALMA 7-m array, revealed an extended dust disk with a peak radius of 140au, probably tracing the location of the planetesimal belt in the system. The disk is outstandingly large compared to known spatially resolved debris disks and also to protoplanetary disks around stars of comparable masses. The dynamical excitation of the belt at this radius is found to be reconcilable with planetary stirring, while self-stirring by large planetesimals embedded in the belt can work only if these bodies form very rapidly, e.g. via pebble concentration. By analyzing the spectral energy distribution we derived a characteristic dust temperature of 43K and a fractional luminosity of 1.1$\times$10$^{-3}$. The latter value is prominently high, we know only four other similarly dust-rich Kuiper-belt analogs within 40pc of the Sun

Quantum kink and its excitations

We show how detailed properties of a kink in quantum field theory can be extracted from field correlation functions. This makes it possible to study quantum kinks in a fully non-perturbative way using Monte Carlo simulations. We demonstrate this by calculating the kink mass as well as the spectrum and approximate wave functions of its excitations. This way of measuring the kink mass has clear advantages over the existing approaches based on creation and annihilation operators or the kink free energy. Our methods are straightforward to generalise to more realistic theories and other defect types.Comment: 21 pages, 11 figures, v2: typos corrected, references adde

One-loop spectroscopy of semiclassically quantized strings: bosonic sector

We make a further step in the analytically exact quantization of spinning string states in semiclassical approximation, by evaluating the exact one-loop partition function for a class of two-spin string solutions for which quadratic fluctuations form a non-trivial system of coupled modes. This is the case of a folded string in the SU(2) sector, in the limit described by a quantum Landau–Lifshitz model. The same applies to the full bosonic sector of fluctuations over the folded spinning string in AdS5 with an angular momentum J in S5. Fluctuations are governed by a special class of fourth-order differential operators, with coefficients being meromorphic functions on the torus, which we are able to solve exactly

The debris disc of HD 131488: bringing together thermal emission and scattered light

This is the final version. Available from Oxford University Press via the DOI in this record. DATA AVAILABILITY: The data underlying this article will be shared on request to the corresponding author. The ALMA and VLT/SPHERE data are publicly available and can be queried and downloaded directly from the ALMA archive: https://almascience.nrao.edu/asax/ and the SPHERE archive: https://archive.eso.org/wdb/wdb/eso/sphere/.We show the first SPHERE/IRDIS and IFS data of the CO-rich debris disc around HD 131488. We use N-body simulations to model both the scattered light images and the spectral energy distribution of the disc in a self-consistent way. We apply the Henyey–Greenstein approximation, Mie theory, and the Discrete Dipole Approximation to model the emission of individual dust grains. Our study shows that only when gas drag is taken into account can we find a model that is consistent with scattered light as well as thermal emission data of the disc. The models suggest a gas surface density of 2 × 10−5 M⊕ au−2 which is in agreement with estimates from ALMA observations. Thus, our modelling procedure allows us to roughly constrain the expected amount of gas in a debris disc without actual gas measurements. We also show that the shallow size distribution of the dust leads to a significant contribution of large particles to the overall amount of scattered light. The scattering phase function indicates a dust porosity of ∼0.2…0.6 which is in agreement with a pebble pile scenario for planetesimal growth.Agence Nationale de la RechercheSwiss National Science Foundation (SNSF)CNR

Water in the terrestrial planet-forming zone of the PDS 70 disk

Terrestrial and sub-Neptune planets are expected to form in the inner ($<10~$AU) regions of protoplanetary disks. Water plays a key role in their formation, although it is yet unclear whether water molecules are formed in-situ or transported from the outer disk. So far Spitzer Space Telescope observations have only provided water luminosity upper limits for dust-depleted inner disks, similar to PDS 70, the first system with direct confirmation of protoplanet presence. Here we report JWST observations of PDS 70, a benchmark target to search for water in a disk hosting a large ($\sim54~$AU) planet-carved gap separating an inner and outer disk. Our findings show water in the inner disk of PDS 70. This implies that potential terrestrial planets forming therein have access to a water reservoir. The column densities of water vapour suggest in-situ formation via a reaction sequence involving O, H$_2$, and/or OH, and survival through water self-shielding. This is also supported by the presence of CO$_2$ emission, another molecule sensitive to UV photodissociation. Dust shielding, and replenishment of both gas and small dust from the outer disk, may also play a role in sustaining the water reservoir. Our observations also reveal a strong variability of the mid-infrared spectral energy distribution, pointing to a change of inner disk geometry.Comment: To appear in Nature on 24 July 2023. 21 pages, 10 figures; includes extended data. Part of the JWST MINDS Guaranteed Time Observations program's science enabling products. Spectra downloadable on Zenodo at https://zenodo.org/record/799102

Identification and characterization of antibacterial compound(s) of cockroaches (Periplaneta americana)

Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential source of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic E. coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analyzed. Among hundreds of compounds, only a few homologous compounds were identified that contained isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole containing analogs, sulfonamides, furanones, flavanones, and known to possess broad-spectrum antimicrobial properties, and possess anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs